Appearances Can Be Deceptive: Revealing a Hidden Viral Infection with Deep Sequencing in a Plant Quarantine Context Thierry Candresse1,2, Denis Filloux3, Brejnev Muhire4, Charlotte Julian3, Serge Galzi3, Guillaume Fort3, Pauline Bernardo3, Jean-Heindrich Daugrois3, Emmanuel Fernandez3, Darren P. Martin4, Arvind Varsani5,6,7, Philippe Roumagnac3* 1 INRA, UMR 1332 Biologie du Fruit et Pathologie, CS 20032, 33882 Villenave d’Ornon Cedex, France, 2 Universite´ de Bordeaux, UMR 1332 Biologie du Fruit et Pathologie, CS 20032, 33882 Villenave d’Ornon Cedex, France, 3 CIRAD, UMR BGPI, Campus International de Montferrier-Baillarguet, 34398 Montpellier Cedex-5, France, 4 Computational Biology Group, Institute of Infectious Diseases and Molecular Medicine, University of Cape Town, Cape Town, South Africa, 5 School of Biological Sciences and Biomolecular Interaction Centre, University of Canterbury, Christchurch, New Zealand, 6 Department of Plant Pathology and Emerging Pathogens Institute, University of Florida, Gainesville, Florida, United States of America, 7 Electron Microscope Unit, Division of Medical Biochemistry, Department of Clinical Laboratory Sciences, University of Cape Town, Observatory, South Africa Abstract Comprehensive inventories of plant viral diversity are essential for effective quarantine and sanitation efforts. The safety of regulated plant material exchanges presently relies heavily on techniques such as PCR or nucleic acid hybridisation, which are only suited to the detection and characterisation of specific, well characterised pathogens. Here, we demonstrate the utility of sequence-independent next generation sequencing (NGS) of both virus-derived small interfering RNAs (siRNAs) and virion-associated nucleic acids (VANA) for the detailed identification and characterisation of viruses infecting two quarantined sugarcane plants. Both plants originated from Egypt and were known to be infected with Sugarcane streak Egypt Virus (SSEV; Genus Mastrevirus, Family Geminiviridae), but were revealed by the NGS approaches to also be infected by a second highly divergent mastrevirus, here named Sugarcane white streak Virus (SWSV). This novel virus had escaped detection by all routine quarantine detection assays and was found to also be present in sugarcane plants originating from Sudan. Complete SWSV genomes were cloned and sequenced from six plants and all were found to share .91% genome- wide identity. With the exception of two SWSV variants, which potentially express unusually large RepA proteins, the SWSV isolates display genome characteristics very typical to those of all other previously described mastreviruses. An analysis of virus-derived siRNAs for SWSV and SSEV showed them to be strongly influenced by secondary structures within both genomic single stranded DNA and mRNA transcripts. In addition, the distribution of siRNA size frequencies indicates that these mastreviruses are likely subject to both transcriptional and post-transcriptional gene silencing. Our study stresses the potential advantages of NGS-based virus metagenomic screening in a plant quarantine setting and indicates that such techniques could dramatically reduce the numbers of non-intercepted virus pathogens passing through plant quarantine stations. Citation: Candresse T, Filloux D, Muhire B, Julian C, Galzi S, et al. (2014) Appearances Can Be Deceptive: Revealing a Hidden Viral Infection with Deep Sequencing in a Plant Quarantine Context. PLoS ONE 9(7): e102945. doi:10.1371/journal.pone.0102945 Editor: Hanu Pappu, Washington State University, United States of America Received January 31, 2014; Accepted June 24, 2014; Published July 25, 2014 Copyright: ß 2014 Candresse et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the ANR French National Research Agency (Netbiome Program, SafePGR Project). AV and DPM are supported by the National Research Foundation of South Africa. BM is funded by the University of Cape Town. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * Email: [email protected] Introduction The major challenge of using classical nucleic acid sequence- informed detection tools such as polymerase chain reaction (PCR) When attempting to prevent the spread of plant diseases, or Southern hybridisation assays, is that despite being highly comprehensive inventories of viral diversity are fundamental both sensitive, these techniques are generally either species or, at best, for effective quarantine and sanitation efforts, and to ensure that genus-specific. In addition, such tools lack the capacity to detect, plant materials within biological resource centres (BRCs) can be let alone identify, pathogens that are unknown, poorly character- safely distributed [1,2]. Detection of pathogens is one of the most ized or highly variable. Although it might be argued that the most critical quarantine and BRC operations. Ideally, the tools used for economically important pathogens tend to be well characterized this purpose must be both sensitive enough to accurately detect the and that it is therefore not a serious issue that many of the more presence of even extremely low amounts of pathogen nucleic acids obscure pathogens go undetected, it is becoming better appreci- or proteins, and provide sufficient specific information to identify ated that the ‘‘importance’’ of any particular pathogenic microbe the genetic variants/strains of whatever pathogens are present. is very difficult to define. Specifically, the environmental and PLOS ONE | www.plosone.org 1 July 2014 | Volume 9 | Issue 7 | e102945 Viral Metagenomics in a Plant Quarantine Context economic impacts of a particular pathogen can vary widely with Materials and Methods varying climatic and ecological conditions and there are large numbers of microbes that are presently not classified as pathogens Plant material and sugarcane quarantine DNAs collection (or at least which are not noticeably pathogenic to humans or to Leaves presenting typical symptoms of sugarcane streak disease domesticated plants and animals), which will eventually emerge as were sampled from two sugarcane plants that had previously been important future pathogens [3]. Also, since non-domesticated found to be infected with Sugarcane streak Egypt virus (SSEV) plant and animal species and countless numbers of microbes which and had been kept in a quarantine greenhouse at the CIRAD contribute to natural terrestrial ecosystems [4–6] can also Sugarcane Quarantine Station, in Montpellier, France. The two potentially be threatened by exotic pathogens, the unconstrained sugarcane plants, VARX and USDA (which was initially global dissemination of apparently harmless fungi, viruses and maintained at USDA-APHIS Plant Germplasm Quarantine bacteria could have serious environmental and economic impacts. before being transferred to CIRAD in 2007), were both initially Whereas ‘‘sequence-dependent’’ microbial detection methods, collected in Egypt during two independent sampling surveys in the which are generally based on PCR or nucleic acid hybridisation, late 1990s [25,26]. These sampling surveys were both carried out can only be used to target known pathogens, sequence-indepen- on experimental stations and commercial lands in close collabo- dent next generation sequencing (NGS) based approaches can ration with Egyptian authorities (Sugar Crop Research Institute potentially provide an ideal platform for identifying almost all (SCRI), Dr Abdel Wahab I. Allam (Director of SCRI) regarding known and unknown microbes present in any particular host VARX; and Agricultural Genetic Engineering Research Institute, organism [5,7–9]. Coupled with innovative sample processing Dr N.A. Abdallah, and Dr M.A. Madkour regarding USDA). In procedures, ‘‘metagenomics’’ applications of NGS [10] have addition, leaves from six sugarcane plants originating from Sudan already enabled the identification of novel pathogens through (B0065, B0067, B0069, D0002, D0003 and D0005, Table S1) and the rapid and comprehensive characterization of microbial strains maintained at the CIRAD Sugarcane Quarantine Station were also used (Material Transfer Agreements between CIRAD and and isolates within environmental and host tissue samples [9,11]. Kenana Sugar Co. Ltd). DNAs from these six plants were In addition to numerous applications in the study of animal extracted using the DNeasy Plant Mini Kit (Qiagen). In addition, infecting viruses, NGS-based metagenomics approaches have also DNA was extracted from an additional 18 frozen leaf samples (2 been used to detect plant infecting viruses [12]. Three main classes 20uC), including 17 samples originating from Sudanese sugarcane of nucleic-acids have been targeted by such analyses: (1) virion- plants, which had passed through the Montpellier Quarantine associated nucleic acids (VANA) purified from viral particles station between 2000 and 2009 and one which had been obtained [13,14]; (2) double-stranded RNAs (dsRNA) [15]; and (3) virus- from a sugarcane seedling grown from sugarcane true seeds [fuzz] derived small interfering RNAs (siRNAs) [16]. Large numbers of developed in Guadeloupe from a biparental cross involving plants both known and new